Bifidobacterium bifidum plantbase-931 for removing dampness and reducing weight, fermentation product thereof, and preparation method and application thereof

Abstract

This invention relates to the field of probiotics technology, specifically to a Bifidobacterium bifidum PlantBase-931 for removing dampness and aiding weight loss, its fermented products, preparation methods, and applications. Bifidobacterium bifidum PlantBase-931 can increase the levels of GAS and MTL in the blood, strengthen the spleen and stomach, promote the expression of AQP3 and AQP4 in colon tissue, and accelerate the elimination of dampness from the body, thus benefiting obese individuals in their weight loss journey. The PlantBase-931 fermented product is prepared by staged fermentation, spray drying, and vacuum freeze-drying of the composition. The composition consists of the following ingredients in parts by weight: navel orange, hawthorn, grape, lychee, papaya, coix seed, winter melon, white sugar, and purified water. The resulting fermented product can reduce IL-1β inflammation levels in the spleen, repair spleen damage, and improve loose stool symptoms in obese individuals.

Description

Technical Field

[0001] This invention relates to the field of probiotics technology, specifically to a Bifidobacterium bifidum PlantBase-931 for removing dampness and aiding weight loss, its fermented products, preparation methods, and applications. Background Technology

[0002] There are many types of obesity. Besides excess fat, excessive dampness can also lead to obesity. This type of obesity is characterized by symptoms such as low food intake but high weight, fatigue, swollen hands and feet, pale complexion, loose muscles, and loose stools. Traditional Chinese medicine believes that the causes of obesity mainly include congenital factors, irregular diet, overwork and lack of rest, and emotional imbalance. Its pathology is often characterized by deficiency in the root and excess in the branch. The root deficiency is mainly spleen and kidney deficiency, while the branch excess is mainly phlegm and dampness, along with blood stasis, heat accumulation, and qi stagnation. Clinically, simple obesity due to spleen deficiency and dampness obstruction is also common.

[0003] The spleen governs transformation and transportation, which includes two aspects: the transformation and transportation of food and water, and the transformation and transportation of fluids. When the spleen's function of transformation and transportation is normal, the "foundation of acquired constitution" is replenished and the "source of qi and blood" is abundant. If the spleen's function of transformation and transportation is impaired, the essence of food and water cannot be absorbed normally, and the metabolism of fluids cannot proceed normally. Phlegm and dampness accumulate in the body, leading to symptoms such as obesity. The kidneys store essence and are the root of yin and yang. Therefore, the qi transformation of the kidneys plays a decisive role in the metabolism of body fluids. If the kidney qi is deficient and the qi transformation is impaired, the body's fluid metabolism will be unbalanced, and the metabolism of phlegm, dampness, and water will be abnormal, which will also lead to obesity.

[0004] Ancient Chinese texts also contain numerous accounts of obesity. The *Book of Rites* states, "When the skin is full and plump, a person is considered obese." The *Huangdi Neijing* was the first to link obesity to constitution, and the *Lingshu* further categorized obese individuals into three types: "fatty," "abdominal fat," and "fleshy." It wasn't until the Song Dynasty that Yang Renzhai formally pointed out the pathological constitution of obese individuals in his *Renzhai Zhifang* (Treatise on the Differentiation of Fire and Dampness): "Obese people have weak qi, which generates cold; cold generates dampness; dampness generates phlegm… therefore, obese people often suffer from cold and dampness."

[0005] Bifidobacterium bifidum is an anaerobic bacterium that primarily colonizes the gut, present even in the intestines of infants during lactation. During its metabolism, it produces various beneficial bioactive substances such as flavonoids, short-chain fatty acids, and extracellular polysaccharides, regulating the positive balance of the gut microbiota, promoting metabolism, and is closely related to human health. It is beneficial and harmless, and remains a lifelong companion. Studies have shown that Bifidobacterium bifidum is the dominant species in the gut of healthy young adults. However, in obese individuals, the number of Bifidobacterium bifidum decreases sharply, leading to gut microbiota dysbiosis. Therefore, Bifidobacterium bifidum with weight-loss capabilities is needed to improve this condition. Different strains exhibit significantly different weight-loss mechanisms and effects due to variations in their gene sequences. Through extensive experiments, we discovered that a single strain of Bifidobacterium bifidum can improve obesity symptoms by influencing the digestion and absorption of food in the esophagus, thereby regulating the spleen and kidneys. Summary of the Invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide Bifidobacterium bifidum PlantBase-931 for removing dampness and losing weight, its fermentation products, preparation methods and applications.

[0007] The present invention provides Bifidobacterium bifidum PlantBase-931, characterized in that the preservation number of Bifidobacterium bifidum PlantBase-931 is GDMCC No.66870.

[0008] In some embodiments, the 16S rDNA gene sequence of the Bifidobacterium plantBase-931 is shown in SEQ ID NO.1.

[0009] Compared with other Bifidobacteria, the strain of this invention can strengthen the spleen and stomach, accelerate the excretion of dampness in the body, and help obese people lose weight.

[0010] The Bifidobacterium plantBase-931 described in this invention exerts its effect of removing dampness and promoting weight loss from at least one of the following (1) to (8):

[0011] (1) Increase blood GAS and MTL levels, and strengthen the spleen and stomach;

[0012] (2) Weight loss and weight gain;

[0013] (3) Increase the relative mRNA expression levels of AQP3 and AQP4 in colon tissue and accelerate the excretion of moisture;

[0014] (4) Increase the polypeptide content in fermented products;

[0015] (5) Increase the content of antioxidant peptides in fermented products;

[0016] (6) Improve the DPPH∙free radical scavenging rate of fermented products;

[0017] (7) Improve the ABTS of fermented products + Free radical scavenging rate;

[0018] (8) Increase the vitamin B6 content in fermented products.

[0019] The present invention also provides a fermented product for removing dampness and losing weight, which includes Bifidobacterium bifidum PlantBase-931 as described above and the composition.

[0020] In some embodiments, the composition comprises the following materials in parts by weight: 10-20 parts navel orange, 1-10 parts hawthorn, 10-20 parts grape, 1-10 parts lychee, 5-15 parts papaya, 1-10 parts coix seed, 1-10 parts winter melon, 5-15 parts white sugar, and 42 parts purified water.

[0021] The components and proportions of the composition were determined through systematic experimental screening and optimization. Compared with other formulations (such as those lacking hawthorn, or replacing some components with oranges or fox nuts), the fermented products prepared using this composition and proportion can reduce weight gain, reduce IL-1β inflammation levels in the spleen, repair spleen damage, reduce the frequency of loose stools, improve loose stool symptoms in obese individuals, and are beneficial for removing dampness and losing weight.

[0022] The specific combination of navel orange, hawthorn, grape, lychee, papaya, coix seed, winter melon, and white sugar used in this application has significant advantages in synergistic effects. Specifically, compared to using tangerines, navel oranges are rich in vitamin C, beta-carotene, organic acids, pectin, olefins, and other substances, which can strengthen the spleen and stomach, aid digestion, and help clear heat and dampness, eliminate weight gain caused by a high-fat diet. Among them, d-limonene can not only act on the smooth muscles of the human body, relaxing them and preventing or reducing stomach cramps, but also help balance stomach acid, further reducing stomach discomfort.

[0023] Compared to gorgon fruit, coix seed, also known as Job's tears, is described in the "Shennong's Classic of Materia Medica" as sweet and cool in nature, with the effects of promoting diuresis and removing dampness, strengthening the spleen and stopping diarrhea, relieving numbness, draining pus, detoxifying and dispersing nodules. In recent years, many studies have reported that coix seed extract can be used as a therapeutic agent to treat obesity. Experimental results show that coix seed can reduce serum cholesterol and triglyceride levels.

[0024] Hawthorn is sour, sweet, and slightly warm in nature. It enters the spleen, stomach, and liver meridians and has the effects of promoting digestion, strengthening the stomach, regulating qi and dispersing blood stasis, and clearing turbidity and lowering lipids. Clinically, it can be used for food stagnation, abdominal distension, diarrhea and abdominal pain, amenorrhea due to blood stasis, postpartum blood stasis, stabbing pain in the heart and abdomen, chest pain, hernia pain, and hyperlipidemia.

[0025] Grapes enter the lung, spleen, and kidney meridians, and can replenish the qi and blood of the spleen and stomach, and nourish the lungs and kidneys. Ye Gui's "Ben Cao Zai Xin" from the Qing Dynasty records that grapes enter the spleen and lung meridians and can "warm the stomach and strengthen the spleen." Huang Gongxiu's "Ben Cao Qiu Zhen" from the Qing Dynasty also records that "(grapes) are sweet, salty, and warm in nature, and can retain essence and qi, and return to the kidneys, and their effects are not far from those of schisandra chinensis." In addition, modern pharmacological studies have found that grapes are also rich in proanthocyanidins (PC) and resveratrol (Res), which can reduce the absorption of cholesterol in the small intestine and have a lipid-lowering effect.

[0026] Lychee is a medicinal herb where "every part is valuable," but from ancient times to the present, the lychee seed, pulp, and shell are the most commonly used parts for medicinal purposes. Lychee pulp can nourish the spleen and blood, invigorate yang and qi, and treat metrorrhagia, spleen deficiency diarrhea, weakness after illness, scrofula, tumors, redness and swelling, etc.

[0027] Papaya, also known as "longevity fruit", is slightly cold in nature and sweet and neutral in taste. It has the effects of strengthening the spleen and stomach, aiding digestion, moisturizing the lungs and relieving dryness, clearing heat and phlegm, promoting lactation, facilitating urination and defecation, preventing skin aging, and removing dark spots and freckles. It also has antibacterial, anti-inflammatory, insecticidal, anti-tinea, and decontamination effects.

[0028] According to the "Shennong's Classic of Materia Medica", winter melon is slightly cold in nature, sweet and bland in taste, and non-toxic. It enters the lung, large intestine, small intestine, and bladder meridians. It has the effects of clearing lung heat, resolving phlegm, clearing stomach heat, relieving irritability and thirst, removing dampness and relieving summer heat, promoting urination, and eliminating edema. In addition, winter melon is rich in malonic acid, which can effectively control the conversion of carbohydrates into fat in the body and prevent the accumulation of fat in the body. It has a good effect on preventing and treating hypertension, arteriosclerosis, and weight loss.

[0029] In summary, the combination of these ingredients complements each other in terms of spleen and stomach care, anti-inflammatory effects, clearing heat and dampness, promoting diuresis and eliminating dampness, and weight loss and fat reduction, laying a solid foundation for the preparation of fermented products with good dampness-removing and weight-loss effects.

[0030] In some embodiments, the composition comprises the following ingredients in parts by weight: 15 parts navel orange, 5 parts hawthorn, 10 parts grape, 5 parts lychee, 8 parts papaya, 3 parts Job's tears, 5 parts winter melon, 7 parts white sugar, and 42 parts purified water. This ratio was determined through experimental optimization. Compared to other ratios, the fermented product prepared from this composition can reduce weight gain, decrease IL-1β inflammation levels in the spleen, repair spleen damage, reduce the frequency of loose stools, improve loose stool symptoms in obese individuals, and is beneficial for removing dampness and losing weight.

[0031] Furthermore, this invention provides a method for preparing a fermented product of Bifidobacterium bifidum PlantBase-931 for removing dampness and promoting weight loss, comprising:

[0032] After soaking, decocting and filtering, coix seed water is obtained. Navel orange, papaya, hawthorn, grape, winter melon and lychee are placed in a high-speed blender, and then white sugar and purified water are added. After blending, the filtrate is filtered out. The filtrate is then mixed with coix seed water and sterilized. After cooling, No. 1 liquid is obtained.

[0033] Bifidobacterium bifidum PlantBase-931 was activated to prepare a seed culture medium; the seed culture medium was added to the No. 1 material solution for staged fermentation, centrifugation, spray drying, vacuum freeze drying, pulverization and mixing to obtain the fermented product.

[0034] In some embodiments, the preparation of the seed culture of Bifidobacterium bifidum PlantBase-931 includes: inoculating Bifidobacterium bifidum PlantBase-931 frozen at -80℃ onto an MRS solid plate, anaerobically culturing at 37℃ for 48 h, then picking a single colony from the solid plate and transferring it to 5 mL of culture medium A, anaerobically culturing at 37℃ for 24 h, activating it three times consecutively, then centrifuging at 4℃ and 6000 r / min for 10 min, collecting the precipitate, and diluting it with material No. 1 to a bacterial concentration of 1×10⁻⁶. 9 CFU / mL was used as the seed culture medium.

[0035] In some embodiments, the conditions for the staged fermentation include: the first stage fermentation involves adding 4-6 parts of seed culture medium to the No. 1 feed solution and anaerobically incubating at 37°C for 0-24 hours; the second stage fermentation involves adding 4-6 parts of seed culture medium to the No. 1 feed solution and anaerobically incubating at 37°C for 0-24 hours. For example, the amount of seed culture medium added in the first stage is 4, 5, or 6 parts, the amount of seed culture medium added in the second stage is 4, 5, or 6 parts, and the fermentation time in the first stage is 0, 8, 16, or 24 hours, and the fermentation time in the second stage is 0, 8, 16, or 24 hours. Specifically, the first stage fermentation involves adding 4 parts of seed culture medium to the No. 1 feed solution and anaerobically incubating at 37°C for 16 hours; the second stage fermentation involves adding 6 parts of seed culture medium to the No. 1 feed solution and anaerobically incubating at 37°C for 8 hours. The fermentation time, after being screened and optimized through experiments, can improve the quality of Bifidobacterium bifidum PlantBase-931 fermented products compared to other conditions. This allows Bifidobacterium bifidum PlantBase-931 to produce abundant antioxidant peptides and prebiotics such as vitamin B6. After consumption, these products can better reduce weight gain, lower IL-1β inflammation levels in the spleen, repair spleen damage, improve loose stools, and promote weight loss by eliminating dampness.

[0036] In some embodiments, the spray drying conditions include an inlet air temperature of 150–200°C; for example, the inlet air temperature is 150, 165, 180, or 200°C. Specifically, the inlet air temperature is 165°C. This inlet air temperature has been experimentally screened and optimized. Compared with other conditions, it can improve the quality of Bifidobacterium bifidum PlantBase-931 fermented products, better protect the vitamin B6 content in the fermented products, and after human consumption, it can better reduce the frequency of loose stools and improve the loose stool condition in obese individuals.

[0037] In some embodiments, the vacuum freeze-drying conditions include: during the second drying process, heating the shelf temperature to 25–35°C within 1 hour; for example, the temperature is 25, 30, or 35°C. Specifically, during the second drying process, heating the shelf temperature to 25°C within 1 hour is optimized through testing. This temperature, compared to other conditions, can improve the quality of Bifidobacterium plantBase-931 fermented products, better protect the viable count of PlantBase-931 in the fermented products, and after human consumption, can better reduce the frequency of loose stools and improve the loose stool condition in obese individuals.

[0038] Furthermore, the present invention also provides Bifidobacterium bifidum PlantBase-931 as described above, Bifidobacterium bifidum PlantBase-931 fermented product as described above, or Bifidobacterium bifidum PlantBase-931 fermented product obtained by the preparation method described above, characterized in that it is used in at least one of the following (1) to (9):

[0039] (1) Application in the preparation of products for removing dampness and losing weight;

[0040] (2) Application in the preparation of dehumidifying products;

[0041] (3) Application in the preparation of weight loss products;

[0042] (4) Application in the preparation of products for strengthening the spleen and stomach;

[0043] (5) Application in the preparation of products that increase blood GAS and MTL levels;

[0044] (6) Application in the preparation of products that increase the relative mRNA expression levels of AQP3 and AQP4 in colon tissue;

[0045] (7) Application in the preparation of products that reduce IL-1β inflammation levels in the spleen;

[0046] (8) Application in the preparation of products for repairing spleen damage;

[0047] (9) Application in the preparation of products that reduce the frequency of loose stools.

[0048] Furthermore, the applications include live bacteria and inactivated bacteria.

[0049] In some embodiments, the dosage form of the product includes solid dosage forms, liquid dosage forms, or semi-solid dosage forms.

[0050] Compared with the prior art, the beneficial effects of the present invention are:

[0051] 1. The Bifidobacterium bifidum PlantBase-931 of the present invention can increase the content of blood GAS and MTL, strengthen the spleen and stomach, and also significantly increase the relative mRNA expression of AQP3 and AQP4 in colon tissue. It can accelerate the excretion of dampness, reduce weight gain, and is beneficial for removing dampness and losing weight.

[0052] 2. The composition of this invention uses carefully selected ingredients such as navel orange, hawthorn, grape, lychee, papaya, coix seed, and winter melon as core components. It is rich in nutrients, containing various vitamins, organic acids, proteins, sugars, and functional components (such as β-carotene, coixol, anthocyanins, and malonic acid). It not only promotes diuresis and eliminates dampness, strengthens the spleen and stomach, and reduces the toxic effects of dampness on the spleen and stomach, but also eliminates weight gain caused by a high-fat diet. The combination of these components works synergistically in terms of spleen and stomach care, anti-inflammatory effects, clearing heat and dampness, promoting diuresis and eliminating dampness, and weight loss, thus aiding in the removal of dampness and weight loss. This invention is highly innovative in terms of its raw material composition alone, and the specific proportions further improve the quality of the product.

[0053] 3. The composition is processed using the phased fermentation, centrifugation, spray drying, vacuum freeze-drying, pulverization, and mixing processes of Bifidobacterium bifidum PlantBase-931 of this invention to obtain a fermented product of Bifidobacterium bifidum PlantBase-931. The resulting fermented product can reduce weight gain, reduce IL-1β inflammation levels in the spleen, repair spleen damage, reduce the frequency of loose stools, improve loose stool symptoms in obese individuals, and significantly enhance the effects of removing dampness and aiding weight loss.

[0054] 4. The Bifidobacterium plantBase-931 fermented product of the present invention creatively adopts a staged fermentation method and a spray drying combined with vacuum freeze drying method during the preparation process. This not only allows the composition to ferment fully during the fermentation stage, achieving effective conversion of nutrients while producing rich antioxidant peptides and probiotic factors such as vitamin B6, but also allows the fermented product to retain vitamin B6 and Bifidobacterium plantBase-931 to a great extent during the drying stage. After a period of consumption, it can remove dampness and promote weight loss.

[0055] Biological Preservation Instructions:

[0056] Bifidobacterium bifidum PlantBase-931 was deposited on August 22, 2025 at the Guangdong Provincial Center for Microbial Culture Collection; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Academy of Sciences Institute of Microbiology; accession number: GDMCC No. 66870. Attached Figure Description

[0057] Figure 1 A flow chart of the preparation process of fermented products from Bifidobacterium bifidum PlantBase-931;

[0058] Figure 2 The results show the determination of polypeptide content in solution C of the fermentation product of Bifidobacterium bifidum PlantBase-931 in Example 5;

[0059] Figure 3 The results of DPPH·free radical scavenging rate determination of solution C of the Bifidobacterium plantBase-931 fermentation product in Example 5;

[0060] Figure 4 ABTS for solution C of the Bifidobacterium plantBase-931 fermentation product from Example 5. + Results of free radical scavenging rate measurement;

[0061] Figure 5 The results show the determination of vitamin B6 (calculated as pyridoxine) content in the fermented product of Bifidobacterium bifidum PlantBase-931 in Example 5. Detailed Implementation

[0062] This invention provides a method for preparing and applying Bifidobacterium bifidum PlantBase-931 for removing dampness and promoting weight loss, its fermented products, and related applications. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0063] Unless otherwise defined in this invention, the scientific and technical terms associated with this invention shall have the meanings understood by one of ordinary skill in the art.

[0064] The terms “comprising,” “including,” and “having” are used interchangeably to indicate the inclusiveness of a scheme, meaning that the scheme may contain elements other than those listed. It should also be understood that the use of “comprising,” “including,” and “having” herein also provides for schemes “consisting of…”.

[0065] When used herein, the term “and / or” includes the meaning of “and,” “or,” and “all or any other combination of elements linked by the term.”

[0066] The term "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0067] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0068] The numerical ranges and parameters involved in this invention have been presented as precisely as possible in the specific embodiments. However, any numerical value inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all numerical ranges or specific data used in this disclosure may have a reasonable deviation within a certain range, such as ±10%, ±5%, ±1%, or ±0.5%.

[0069] In order to overcome the shortcomings of the existing technology, one of the objectives of this invention is to provide a Bifidobacterium bifidum PlantBase-931 that can remove dampness and promote weight loss, which can strengthen the spleen and stomach, accelerate the excretion of dampness in the body, and help obese people lose weight.

[0070] The second objective of this invention is to provide a method for preparing a fermented product of Bifidobacterium bifidum PlantBase-931 for removing dampness and promoting weight loss. The composition is fermented using Bifidobacterium bifidum PlantBase-931, and the resulting fermented product contains abundant antioxidant peptides, vitamin B6 and Bifidobacterium bifidum PlantBase-931, which can repair spleen damage and improve the symptoms of loose stools in obese people.

[0071] One of the objectives of this invention is achieved through the following technical solution:

[0072] PlantBase-931, a strain of Bifidobacterium that promotes weight loss and eliminates dampness, was obtained through screening via physicochemical testing and animal experiments.

[0073] Strains extracted:

[0074] 1. Preparation of solid culture medium: 10.0 g / L peptone, 10.0 g / L beef extract, 5.0 g / L yeast extract, 20.0 g / L glucose, 1.0 mL / L Tween 80, 2.0 g / L K2HPO4∙7H2O, 5.0 g / L sodium acetate∙3H2O, 2.0 g / L triammonium citrate, 0.1 g / L MgSO4∙7H2O, 0.05 g / L MnSO4∙4H2O, 15.0 g / L agar powder, 15.0 g / L calcium carbonate, add purified water to make up to volume, heat to dissolve, adjust pH to 6.5, autoclave at 121℃ for 20 min, cool to 45℃ to obtain solution A. Using a sterile syringe, the mupirocin lithium salt stock solution and cysteine ​​hydrochloride stock solution were added to solution A to make the concentration of mupirocin lithium salt in the culture medium 50 μg / mL and the concentration of cysteine ​​hydrochloride 500 μg / mL. After shaking well, the solution was poured into plates for later use.

[0075] 2. Take 1g of fresh feces from a healthy vegetarian adult, mix it with 10mL of sterile physiological saline, and then spread 10μL onto a solid culture medium. Incubate anaerobically at 37℃ for 48h. Pick one colony and continue to streak to purify it. Finally, a pure strain was obtained. The 16S rDNA was amplified, and the amplified product was sequenced to identify the strain as Bifidobacterium bifidum, which was named PlantBase-931.

[0076] PlantBase-931 gene sequence identification results:

[0077] CGCGGCGTTGCTGATCCGCGATTACTAGCGACTCCGCCTTCACGGAGCCGGGTTGCAGGCTCCGATCCGAACTGAGACCGGTTTTCAGGGATCCGCTCCATGTCGCCATGTCGCATCCCGCTGTACCGGCCATTGTAGCATGCGTGAAGCCCTGGACGTAAGGGGCATGATGATCTGACGTCATCCCCACCTTCCTCCGAGTTAACCCCGGCGGTCCCCCGTGAGTTCCCACCATAACGTGCTGGCAACACGGGGCGAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACGACCATGCACCACCTGTGAACCCGCCCCGAAGGGAAACGCCATCTCTGGCGTCGTCGGGAACATGTCAAGCCCAGGTAAGGTTCTTCGCGTTGCATCGAATTAATCCGCATGCTCCGCCGCTTGTGCGGGCCCCCGTCAATTTCTTTGAGTTTTAGCCTTGCGGCCGTACTCCCCAGGCGGGACGCTTAACGCGTTAGCTCCGACACGGAACACGTGGAACGTGCCCCACATCCAGCGTCCACCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTCGCTCCCCACGCTTTCGCTCCTCAGCGTCAGTGACGGCCCAGAGACCTGCCTTCGCCATCGGTGTTCTTCCCGATATCTACACATTCCACCGTTACACCGGGAATTCCAGTCTCCCCTACCGCACTCCAGCCCGCCCGTACCCGGCGCAGATCCACCGTTAAGCGATGGACTTTCACACCGGACGCGACGAGCCGCCTACGAGCCCTTTACGCCCAATAAATCCGGATAACGCTTGCGCCCTACGAATTACCGCGGCTGCTGC (SEQ ID NO.1)

[0078] The second object of the present invention is achieved by the following technical solutions:

[0079] A fermented product containing Bifidobacterium bifidum PlantBase-931 for removing dampness and promoting weight loss is prepared by a combination of probiotics through a staged fermentation, spray drying, and vacuum freeze drying process. The probiotic is Bifidobacterium bifidum PlantBase-931. The composition consists of the following ingredients in parts by weight: 15 parts navel orange, 5 parts hawthorn, 10 parts grape, 5 parts lychee, 8 parts papaya, 3 parts coix seed, 5 parts winter melon, 7 parts white sugar, and 42 parts purified water.

[0080] The composition of this invention uses carefully selected ingredients such as navel orange, hawthorn, grape, lychee, papaya, coix seed, and winter melon as core components. It is rich in nutrients, containing various vitamins, organic acids, proteins, sugars, and functional components (such as β-carotene, coixol, anthocyanins, and malonic acid). It not only promotes diuresis and eliminates dampness, strengthens the spleen and stomach, and reduces the toxic effects of dampness on the spleen and stomach, but also eliminates weight gain caused by a high-fat diet. These combined ingredients work synergistically in terms of spleen and stomach care, anti-inflammatory effects, clearing heat and dampness, promoting diuresis and eliminating dampness, and weight loss, thus aiding in the elimination of dampness and weight loss. The composition itself is highly innovative, and the specific proportions further improve product quality. The composition is processed using the Bifidobacterium plantBase-931 staged fermentation, centrifugation, spray drying, vacuum freeze-drying, pulverization, and mixing processes of this invention to obtain the fermented product. The preparation process creatively employs a staged fermentation method and a combined spray drying and vacuum freeze drying method. This not only ensures thorough fermentation of the composition during the fermentation stage, achieving effective conversion of nutrients while producing abundant antioxidant peptides and probiotics such as vitamin B6, but also allows for significant retention of vitamin B6 and Bifidobacterium plantBase-931 during the drying stage. The resulting fermented product can reduce weight gain, decrease IL-1β inflammation levels in the spleen, repair spleen damage, reduce the frequency of loose stools, improve loose stool symptoms in obese individuals, and significantly enhance the effects of removing dampness and promoting weight loss.

[0081] The test materials used in this invention are all common commercial products and can be purchased on the market.

[0082] The present invention will be further illustrated below with reference to the embodiments:

[0083] Example 1: Preparation of Bifidobacterium plantBase-931 fermentation product

[0084] 1. Select fresh, mold-free, insect-free, and intact navel oranges, hawthorns, grapes, lychees, papayas, and winter melons; dried, mold-free, and insect-free Job's tears; and qualified white sugar.

[0085] 2. After washing the navel oranges and papayas with clean water and draining them, peel and remove the seeds, weigh the pulp. The weight portions are 15 parts for navel oranges and 8 parts for papayas. After washing the hawthorns, grapes, and winter melons with clean water and draining them, remove the seeds, weigh the pulp (with peel), and the weight portions are 5 parts for hawthorns, 10 parts for grapes, and 5 parts for winter melons. After washing the lychees with clean water and draining them, peel and remove the pits, weigh the pulp, and the weight portion is 5 parts. After washing the Job's tears with clean water and draining them, weigh 3 parts.

[0086] 3. Soak Job's tears in 15 parts purified water for 2 hours, boil and keep for 40 minutes, filter through an 80-mesh filter, and collect the filtrate as Job's tears water.

[0087] 4. Place navel oranges, papayas, hawthorns, grapes, winter melons, and lychees in a high-speed blender, add 7 parts white sugar and 27 parts purified water, blend at 26000 rpm for 2 minutes, and then pass them through 60-mesh, 80-mesh, and 100-mesh filters in sequence. Collect the filtrate, mix the collected filtrate with Job's tears water evenly, keep it at 95℃ for 5 minutes, and then cool it to 37℃ to obtain the No. 1 liquid.

[0088] 5. Prepare a mixed solution with the following concentrations: 20 g / L glucose, 10 g / L peptone, 10 g / L beef extract, 5 g / L yeast extract, 2 g / L sodium acetate, 2 g / L K2HPO4, 2 g / L diammonium citrate, 0.1 g / L MgSO4·7H2O, 0.05 g / L MnSO4·H2O, 1 mL / L L-ween 80, and 1 g / L cysteine ​​hydrochloride. Adjust the pH to 6.8, sterilize at 121℃ for 15 min, and cool to 37℃ to obtain culture medium A.

[0089] 6. PlantBase-931 of *Bifidobacterium bifidum*, frozen at -80℃, was inoculated onto MRS agar plates and anaerobically cultured at 37℃ for 48 h. Single colonies were then picked from the agar plates and transferred to 5 mL of medium A, and anaerobically cultured at 37℃ for 24 h. This activation process was repeated three times. The culture was then centrifuged at 4℃ and 6000 rpm for 10 min, and the precipitate was collected and diluted with solution No. 1 to a bacterial concentration of 1×10⁻⁶. 9 CFU / mL was used as the seed culture medium.

[0090] 7. Add 4 parts of seed culture medium to the No. 1 material solution, and culture anaerobically at 37℃ for 16 hours. After the culture is completed, centrifuge at 4℃, 6000r / min for 10min to separate the precipitate and supernatant, which are denoted as precipitate A and supernatant B, respectively.

[0091] 8. Supernatant B is spray-dried with an inlet air temperature of 165℃, an inlet liquid flow rate of 2 mL / min, and an air velocity of 3 m / min. 3The gas pressure was 0.1 MPa, and the powder was collected as raw material powder No. 1.

[0092] 9. Add 6 parts of seed culture medium to the No. 1 solution and anaerobic culture at 37℃ for 8 hours to obtain the No. 2 solution.

[0093] 10. Add precipitate A to solution No. 2 and stir well. Place the solution in a vacuum freeze dryer for freeze drying. First, lower the temperature of the drying plates to -4℃ within 10 minutes, then pre-freeze to -45.5℃ and maintain this temperature for 1 hour. During the first drying, heat the drying plates to -30℃ within 1 hour and run under a vacuum of 20 Pa for 18 hours. During the second drying, heat the drying plates to 25℃ within 1 hour and run under a vacuum of 2 Pa for 16 hours to obtain the freeze-dried raw material. Grind the freeze-dried raw material to 80 mesh using a pulverizer and collect the powder as raw material powder No. 2.

[0094] 11. Add raw material powder No. 1 to raw material powder No. 2 and mix well to obtain Bifidobacterium plantBase-931 fermentation product.

[0095] Example 2: Screening for bacteria that strengthen the spleen and stomach

[0096] 1. Experimental bacterial strains

[0097] Lactobacillus acidophilus S1 (GDMCC No. 1.5917), Bifidobacterium animalis subsp. lactis Y6-byj (GDMCC No. 1.5923), Lactobacillus paracasei K9-byj (GDMCC No. 1.5918), Bifidobacterium bifidum PlantBase-931, and Lactobacillus plantarum Orange-01 (GDMCC No. 1.4958).

[0098] 2. Preparation of test solution

[0099] (1) Solution A: Weigh 600g of raw rhubarb, add 500mL of distilled water and soak for 1h, decoct for 20min, filter, add 400mL of distilled water to the residue and decoct for 15min, filter, combine the filtrates, concentrate in water bath to 300mL to obtain solution A.

[0100] (2) MRS medium: Prepare MRS medium according to GB 4789.35, adjust pH to 6.5, autoclave at 121℃ for 20 min, and store at 4℃.

[0101] (3) Bacterial culture: The strain was inoculated into MRS medium and anaerobically incubated at 37°C for 48 h, then inoculated again into MRS medium and anaerobically cultured for 18 h. Subsequently, the culture was centrifuged at 4°C and 8000 r / min for 10 min, and the precipitated bacterial cells were resuspended with physiological saline to adjust the viable bacterial concentration to 4.5 × 10⁻⁶. 8 CFU / mL.

[0102] 3. Laboratory animals

[0103] (1) Fifty-six healthy male SD rats, weighing 200-220g, were randomly divided into 7 groups of 8 rats each, as shown in Table 1. Except for group A, which was administered physiological saline by gavage, the other groups were administered solution A by gavage twice a day, 2 mL each time, for 14 consecutive days. From day 15 onwards, each group was administered the drug by gavage. The types of samples administered by gavage are shown in Table 1. The gavage dose was 1 mL / 100g of rat body weight, for 14 consecutive days.

[0104]

[0105] (2) One hour after the last gavage, the eyeball was removed and 2 mL of blood was collected in a dry test tube and centrifuged at 3500 r / min for 15 min at 4 °C. Another 2 mL of blood was collected in a test tube containing 30 μL each of 10% EDTA-2Na and aprotinin and mixed thoroughly. The mixture was centrifuged at 3500 r / min for 10 min at 4 °C. The serum and plasma were collected and stored at -20 °C. Gastrin (GAS) and motilin (MTL) were measured according to the kit method. The results are shown in Table 2.

[0106] 4. Results and Analysis

[0107]

[0108] A decrease in serum GAS can lead to gastrointestinal dysfunction, affecting the spleen's function of transporting and transforming food, resulting in spleen deficiency. A decrease in plasma MTL levels can cause impaired digestion, absorption, and absorption by the spleen and stomach, leading to insufficient production of qi and blood, and symptoms of spleen deficiency. From the perspective of traditional Chinese medicine, poor digestion and insufficient production of qi and blood result in weak qi propulsion, hence the slowed gastrointestinal motility—all symptoms of spleen deficiency.

[0109] As shown in Table 2, compared with group A, the GAS and MTL levels in group B rats were significantly lower, indicating that the rats had gastrointestinal dysfunction and the spleen deficiency rat model was successfully established. Compared with group B, the GAS and MTL levels in groups C to G rats were all increased, indicating that the spleen and stomach function of the rats was improved. That is, probiotics have a spleen-strengthening effect on rats, and different strains have different effects on strengthening the spleen and stomach. Among them, group F had the highest GAS and MTL levels, which were closest to those in group A, indicating that the spleen and stomach function of group F rats was restored best. That is, the Bifidobacterium bifidum PlantBase-931 in group F had the best spleen-strengthening effect. Therefore, Bifidobacterium bifidum PlantBase-931 was selected as the best strain for strengthening the spleen and stomach.

[0110] Example 3: Screening for the strain with the best effect in removing dampness and promoting weight loss

[0111] 1. Experimental strains

[0112] Bifidobacterium bifidum CICC 10395 (accession number: CICC 10395), Bifidobacterium bifidum CICC 6169 (accession number: CICC 6169), Bifidobacterium bifidum CICC 6071 (accession number: CICC 6071), Bifidobacterium bifidum PlantBase-931

[0113] 2. Sample preparation

[0114] (1) Culture medium B: peptone 10.0 g / L, beef extract 10.0 g / L, yeast extract 5.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, K2HPO4∙7H2O 2.0 g / L, sodium acetate∙3H2O 5.0 g / L, triammonium citrate 2.0 g / L, MgSO4∙7H2O 0.1 g / L, MnSO4∙4H2O 0.05 g / L, agar powder 15.0 g / L, calcium carbonate 15.0 g / L, add purified water to make up to volume, heat to dissolve, adjust pH to 6.5, autoclave at 121℃ for 20 minutes. After cooling to 45°C, solution A is obtained. Using a sterile syringe, cysteine ​​hydrochloride stock solution is added to solution A to make the concentration of cysteine ​​hydrochloride in the culture medium 500 μg / mL. After shaking well, it is cooled to 37°C for later use.

[0115] (2) Bacterial suspension: The strain was inoculated into culture medium B and anaerobically incubated at 37°C for 48 h, then inoculated again into culture medium B and anaerobically cultured for 18 h. Subsequently, the bacterial cells were centrifuged at 4°C and 8000 r / min for 10 min, and the precipitated bacterial cells were resuspended with physiological saline to adjust the viable bacterial concentration to 4.5 × 10⁻⁶. 8 CFU / mL.

[0116] 3. Experimental animals: 56 SPF-grade male SD rats, 6 weeks old and weighing 200-220g, were used. All rats were acclimatized for 7 days with free access to food and water. The animal room temperature was 25℃ and the relative humidity was 65%. A 12-hour light and dark cycle was used to simulate day and night.

[0117] 4. Establishment of a Spleen-Deficiency and Dampness-Retention Type Obese Rats: After the adaptation period, the rats were randomly divided into 7 groups, numbered H to N, with 8 rats in each group. Group H was fed a normal purified diet for 56 consecutive days. Groups I to N were fed a high-fat, low-protein, and low-choline diet for 56 consecutive days, with weighted swimming once a day at 9:30 am (the weight of the weighted object was 10% of the body weight, and the water temperature was 20℃). On day 29, the rats were gavaged with lard (2 mL / rat) on odd days and with 4℃ cold water (20 mL / kg) on ​​even days, and were provided with wet bedding for 14 consecutive days. From day 43 to day 56, the rats were fed a high-fat, low-protein, and low-choline diet.

[0118] 5. After modeling, all rats were administered samples by gavage, with each group receiving one type of sample. The corresponding sample types and dosages are shown in Table 3, and this was repeated for 30 consecutive days. On day 1 and day 31, the rats were weighed, and their weight gain was calculated. The average weight gain was calculated, and the results are shown in Table 4. On day 31, the rats' body length (from nose tip to anus) was measured, and Lee's index was calculated. The average weight gain was calculated, and the results are shown in Table 4.

[0119]

[0120]

[0121] 6. After weighing and measuring body length, the animal was euthanized. 40 mg of colon tissue was placed in a 1.5 mL enzyme-free EP tube. The tissue was minced on ice using sterile scissors, and then 500 μL of RNA lysis buffer was added. The tissue was homogenized using an ultrasonic homogenizer, and then 500 μL of RNA dilution buffer was added. The mixture was stirred and allowed to stand for 3 min, followed by a 70°C water bath for 3 min to improve yield. The mixture was centrifuged at 15000 rpm / min for 5 min at 4°C. The supernatant was collected and placed in an EP tube. Anhydrous ethanol (1 / 2 volume of supernatant) was added, and the mixture was pipetted and mixed. The mixture was then transferred to a centrifuge column and centrifuged at 15000 rpm / min for 1 min at 4°C. The filtrate was discarded. 600 μL of RNA wash buffer was added to the centrifuge column and centrifuged at 15000 rpm / min for 1 min. The filtrate was discarded. 50 μL of freshly prepared DNase I incubation solution was added to the centrifuge column and allowed to stand at room temperature for 15 min. Transfer 600 μL of RNA wash buffer to a centrifuge column, centrifuge at 15000 rpm for 1 min, and discard the filtrate. Transfer another 600 μL of RNA wash buffer to the centrifuge column, centrifuge at 15000 rpm for 1 min, and discard the filtrate. Place the centrifuge column on a collection tube and centrifuge at 15000 rpm for 2 min. Transfer the centrifuge column to an elution tube, pipette 50 μL of nuclease-free water to the center of the column membrane, let stand for 2 min, centrifuge at 15000 rpm for 2 min, and collect the precipitate, which is designated as precipitate A. Measure the mRNA expression levels of AQP3 and AQP4 in precipitate A, which represent the relative mRNA expression levels of AQP3 and AQP4 in rat colon tissue, and calculate the average values. The results are shown in Table 4.

[0122] Methods for determining the mRNA expression levels of AQP3 and AQP4: Prepare a reverse transcription mixture. Remove the cDNA template, primers, and amplification kit from the -20℃ freezer, thaw on ice, mix well, and prepare the RT-PCR reaction system. Add 20 μL of the prepared reaction system to an eight-tube array, briefly centrifuge, and then place in a Bio-Rad real-time quantitative PCR instrument for amplification. PCR reaction conditions: 95℃ pre-denaturation for 15 min, 95℃ denaturation for 10 s, 60℃ annealing extension for 30 s, for 40 cycles. Read the Ct value and calculate the fold change in gene expression between the experimental group and the blank control group, which is the relative mRNA expression level. The primer sequences for AQP3 are: upstream CGTGGTTCCGTGGCTCAAGTG, downstream GATGGCAAGGGTGACAGCGAAG. The primer sequences for AQP4 are: upstream GAAGGCGGTCACAGCAGAGTTC, downstream AGTCCAAAGCAGAGGGAGATGAGG.

[0123] 7. Results and Analysis

[0124]

[0125] The degree of obesity in each group of rats was assessed by detecting weight gain and Lee's index. The level of water metabolism was measured by detecting the expression of AQP3 and AQP4 in colon tissue to assess the degree of dampness resistance in each group of rats. Low expression of AQP3 can cause reduced water reabsorption by the colonic mucosa, leading to the accumulation of dampness in the body, diarrhea or loose stools. Reduced expression of AQP4 in the colon can lead to water metabolism disorder in the colon, which is an important factor in the accumulation of dampness in the body and the occurrence and development of diarrhea.

[0126] As shown in Table 4, compared with group H, group I rats showed significantly increased weight gain and Lee's index, and significantly decreased relative mRNA expression levels of AQP3 and AQP4, indicating that group I rats were significantly obese with disordered water metabolism, and the model of spleen deficiency and dampness retention type obesity was successfully established. Compared with group I, group K to N rats showed decreased weight gain and Lee's index, and increased relative mRNA expression levels of AQP3 and AQP4, with varying degrees of increase and decrease. Among them, group N rats showed the lowest weight gain and Lee's index, and the highest relative mRNA expression levels of AQP3 and AQP4, indicating that group N rats showed the best improvement in obesity, faster dampness elimination, and gradual restoration of water metabolism to normal levels. This further demonstrates that Bifidobacterium bifidum PlantBase-931 can accelerate the elimination of dampness in the body, which is beneficial for weight loss in obese individuals.

[0127] Example 4: Effects of Formula Changes on Fermentation Products

[0128] 1. Comparative Example 1: The preparation method of Comparative Example 1 is the same as that of Example 1, except that the weight parts of navel orange in Comparative Example 1 are 5 parts and the weight parts of winter melon are 15 parts. That is, step 2 of Comparative Example 1 is modified to: "After washing the navel orange and papaya with clean water, drain them, peel and remove the seeds, and weigh the pulp, with a weight part of 5 parts of navel orange and 8 parts of papaya; after washing the hawthorn, grape and winter melon with clean water, drain them, remove the seeds, and weigh the pulp (with peel), with a weight part of 5 parts of hawthorn, 10 parts of grape and 15 parts of winter melon; after washing the lychee with clean water, drain it, peel and remove the pit, and weigh the pulp, with a weight part of 5 parts; after washing the coix seed with clean water, drain it and weigh 3 parts."

[0129] 2. Comparative Example 2: The preparation method of Comparative Example 2 is the same as that of Example 1, except that the weight parts of hawthorn in Comparative Example 2 are 0 parts and the weight parts of coix seed are 8 parts. That is, step 2 of Comparative Example 2 is modified to: "After washing the navel orange and papaya with clean water, drain them, peel and remove the seeds, and weigh the pulp, with the weight parts being 15 parts of navel orange and 8 parts of papaya; after washing the hawthorn, grape and winter melon with clean water, drain them, remove the seeds, and weigh the pulp (with peel), with the weight parts being 0 parts of hawthorn, 10 parts of grape and 5 parts of winter melon; after washing the lychee with clean water, drain it, remove the shell and pit, and weigh the pulp, with the weight parts being 5 parts; after washing the coix seed with clean water, drain it and weigh 8 parts."

[0130] 3. Comparative Example 3: The preparation method of Comparative Example 3 is the same as that of Example 1, except that the amount of seed culture medium added is different. Specifically, step 7 of Comparative Example 3 is modified to "Add 6 parts of seed culture medium to the No. 1 solution, anaerobic culture at 37°C for 16 hours, and centrifuge after the culture is completed. The centrifugation conditions are 4°C, 6000 r / min, and centrifugation time is 10 min. Separate the precipitate and the supernatant, and record them as precipitate A and supernatant B, respectively." Step 9 is modified to "Add 4 parts of seed culture medium to the No. 1 solution, anaerobic culture at 37°C for 8 hours to obtain No. 2 solution."

[0131] 4. Comparative Example 4: The preparation method of Comparative Example 4 is the same as that of Example 1, except that the "navel orange" in Comparative Example 4 is replaced with "tangerine".

[0132] 5. Comparative Example 5: The preparation method of Comparative Example 5 is the same as that of Example 1, except that "Coix seed" in Comparative Example 5 is replaced with "Euryale ferox".

[0133] 6. Preparation of suspension: Weigh 1g of Bifidobacterium bifidum PlantBase-931 fermentation product, add it to 8mL of purified water at 37℃, and stir thoroughly until it is in suspension state.

[0134] 7. Sixty-four SPF-grade male SD rats, 6 weeks old and weighing 200–220 g, were selected. All rats were acclimatized for 7 days with free access to food and water. The animal room temperature was 25°C and the relative humidity was 65%, with 12 hours of light and darkness to simulate day and night. The rats were randomly divided into 8 groups of 8 rats each, labeled O, P, Q, R, S, T, U, and V. Group O rats were fed a standard diet, while the other groups were fed a high-fat diet for 60 consecutive days to establish an obese rat model. After modeling, all rats were fed a standard diet and administered gavage at 9:30 AM daily. Rats in groups O and P were gavaged with purified water; rats in group Q were gavaged with the suspension from Example 1; rats in group R were gavaged with the suspension from Comparative Example 1; rats in group S were gavaged with the suspension from Comparative Example 2; rats in group T were gavaged with the suspension from Comparative Example 3; rats in group U were gavaged with the suspension from Comparative Example 4; and rats in group V were gavaged with the suspension from Comparative Example 5. The daily gavage dose was 4 mL for 30 consecutive days. On day 31, the rats were weighed, and the average value for each group was calculated. The results are shown in Table 5. Simultaneously, fresh feces excreted by the rats that day were collected, and the fecal moisture content was determined according to Method 1 of GB 5009.3. The average value for each group was calculated, and the results are shown in Table 5. The rats were then euthanized, and the spleens were quickly collected. The blood on the spleen tissue was first washed off with PBS, and then excess residual water was absorbed with absorbent paper. 0.1g of spleen was then weighed, and PBS was added to prepare a 10% spleen tissue homogenate using a tissue homogenizer. The homogenate was centrifuged at 3000r / min for 10min using a refrigerated centrifuge. The spleen supernatant was aspirated and transferred to a new sterile enzyme-free EP tube, which was stored at -80℃ for later use.

[0135] 8. Using ELISA, the IL-1β content in the spleen tissue of rats was detected according to the corresponding steps in the instructions of the kit. The OD value was read at 450nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the IL-1β content was calculated. The average value of each group was calculated, and the results are shown in Table 5.

[0136] 9. Results and Analysis

[0137]

[0138] As shown in Table 5, compared with group O, rats in group P showed significantly increased weight and fecal moisture content, as well as significantly increased IL-1β levels in the spleen. This indicates that after being fed a high-fat diet, the rats exhibited obesity symptoms, increased splenic inflammation, and exacerbated loose stools. Compared with group P, rats in groups Q to V showed decreased weight and fecal moisture content, as well as decreased IL-1β levels in the spleen, but the degree of decrease varied. This indicates that changes in the formulation affected the quality of the Bifidobacterium plantBase-931 fermented product. Among them, group Q rats had the lowest weight and fecal moisture content, as well as the lowest IL-1β levels in the spleen. This indicates that group Q rats showed the best improvement in obesity symptoms, significantly reduced splenic inflammation, well-repaired spleen, and significantly improved loose stools.

[0139] Example 5: The effect of changes in fermentation time on fermented products

[0140] 1. Comparative Example 6: The preparation method of Comparative Example 6 is the same as that of Example 1, except that the fermentation time of Comparative Example 6 is different. Specifically, step 7 of Comparative Example 6 is modified to "add 4 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37°C for 8 hours, and centrifuge after the culture is completed. The centrifugation conditions are 4°C, 6000 r / min, and centrifugation time is 10 min. Separate the precipitate and the supernatant, and record them as precipitate A and supernatant B, respectively." Step 9 is modified to "add 6 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37°C for 16 hours to obtain material solution No. 2".

[0141] 2. Comparative Example 7: The preparation method of Comparative Example 7 is the same as that of Example 1, except that the fermentation time of Comparative Example 7 is different. Specifically, step 7 of Comparative Example 7 is modified to "add 4 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37°C for 24 hours, and centrifuge after the culture is completed. The centrifugation conditions are 4°C, 6000 r / min, and centrifugation time is 10 min. Separate the precipitate and the supernatant, and record them as precipitate A and supernatant B, respectively." Step 9 is modified to "add 6 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37°C for 0 hours to obtain material solution No. 2".

[0142] 3. Comparative Example 8: The preparation method of Comparative Example 8 is the same as that of Example 1, except that the fermentation time of Comparative Example 8 is different. Specifically, step 7 of Comparative Example 8 is modified to "Add 4 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37℃ for 0 hours, and centrifuge after the culture is completed. The centrifugation conditions are 4℃, 6000r / min, and centrifugation time is 10min. Separate the precipitate and the supernatant, and record them as precipitate A and supernatant B, respectively." Step 9 is modified to "Add 6 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37℃ for 24 hours to obtain material solution No. 2."

[0143] 4. Comparative Example 9: The preparation method of Comparative Example 9 is the same as that of Example 1, except that the fermentation time of Comparative Example 9 is 0 hours. Specifically, step 7 of Comparative Example 9 is modified to "Add 4 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37℃ for 0 hours, and centrifuge after the culture is completed. The centrifugation conditions are 4℃, 6000r / min, and centrifugation time is 10min. Separate the precipitate and the supernatant, and record them as precipitate A and supernatant B, respectively." Step 9 is modified to "Add 6 parts of seed culture medium to the No. 1 material solution, anaerobic culture at 37℃ for 0 hours to obtain material solution No. 2."

[0144] 5. Preparation of solution C: Weigh 2g of Bifidobacterium plantBase-931 fermentation product, add 20mL of purified water, stir well, centrifuge at 6000r / min for 15min, collect the supernatant, pass the supernatant through a polyethersulfone membrane with a pore size of 3kDa, collect the filtered solution, and record it as solution C.

[0145] 6. According to the detection method in Appendix B of GB / T 22492, the content (g / 100g) of polypeptide in solution C of Examples 1, 6, 7, 8, and 9 was detected. The results are shown in Table 6. Figure 2 As shown.

[0146] 7. According to the test method of GB / T 39100, the DPPH·free radical scavenging rate (%) and ABTS of solution C of Examples 1, 6, 7, 8, and 9 were tested. + Free radical scavenging rate (%), the results are shown in Table 6. Figure 3 and Figure 4 As shown.

[0147] 8. According to the detection method of GB 5009.154, the content of vitamin B6 (calculated as pyridoxine) in the fermented products of Bifidobacterium bifidum PlantBase-931 in Examples 1, 6, 7, 8, and 9 was detected. The results are shown in Table 6 and... Figure 5 As shown.

[0148] 9. Preparation of suspension: Weigh 1g of Bifidobacterium bifidum fermentation product, add it to 8mL of purified water at 37℃, and stir thoroughly until it is in a suspended state.

[0149] 10. Thirty-two SPF-grade male SD rats, 6 weeks old and weighing 200–220 g, were selected. All rats were acclimatized for 7 days with free access to food and water. The animal room temperature was 25°C, relative humidity 65%, and a 12-hour light-dark cycle simulated day and night. The rats were randomly divided into four groups of eight, designated W1, W2, W3, and W4. Each group was fed a high-fat diet for 60 consecutive days to establish an obese rat model. After modeling, all rats were fed a standard diet and administered a gavage treatment at 9:30 AM daily. Group W1 received a suspension of control ratio 6, Group W2 received a suspension of control ratio 7, Group W3 received a suspension of control ratio 8, and Group W4 received a suspension of control ratio 9. The daily gavage dose was 4 mL for 30 consecutive days. On day 31, the rats were weighed, and the average weight for each group was calculated. The results are shown in Table 7. Meanwhile, fresh feces excreted by rats on the same day were collected, and the water content of the feces was determined according to the first method of GB 5009.3. The average value of each group was calculated, and the results are shown in Table 7. Subsequently, the rats were euthanized, and the spleen was quickly collected. The blood on the spleen tissue was first washed off with PBS, and then excess residual water was absorbed with absorbent paper. Then, 0.1g of spleen was weighed, added to PBS, and a 10% spleen tissue homogenate was prepared using a tissue homogenizer. The homogenate was centrifuged at 3000r / min for 10min using a refrigerated centrifuge. The spleen supernatant was aspirated and transferred to a new sterile enzyme-free EP tube and stored at -80℃ for later use.

[0150] 11. Using ELISA, the IL-1β content in the spleen tissue of rats was detected according to the corresponding steps in the kit instructions. The OD value was read at 450 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the IL-1β content was calculated. The average value of each group was calculated, and the results are shown in Table 7.

[0151] 12. Results and Analysis

[0152] (1) Component detection of fermented products

[0153]

[0154] Antioxidant peptides are natural antioxidants secreted during lactic acid bacteria fermentation. They can repair damage to the spleen and other organs, and help improve metabolic disorders, reduce dampness, and promote weight loss. Vitamin B6, as an important vitamin for human metabolic activities, can regulate the normal functioning of metabolic reactions in the body.

[0155] From Table 6, Figures 2-5 It can be seen that, compared with Comparative Examples 6 to 9, Example 1 showed a significant increase in peptide content, DPPH free radical scavenging rate, and ABTS. +The significantly increased free radical scavenging rate indicates that the content of antioxidant peptides in the fermented product of Example 1 is significantly increased. That is, the fermentation process of Example 1 enables Bifidobacterium bifidum PlantBase-931 to produce more antioxidant peptides, which can better remove dampness and promote weight loss. Compared with Comparative Examples 6 to 9, the content of vitamin B6 (calculated as pyridoxine) in Example 1 is significantly increased. The fermentation process of Example 1 enables Bifidobacterium bifidum PlantBase-931 to produce more vitamin B6, which can better regulate metabolic reactions in the human body.

[0156] (2) Obese rat model experiment

[0157]

[0158] As shown in Tables 5 and 7, compared with group Q, the rats in groups W1, W2, W3, and W4 had increased weight and fecal water content, and increased IL-1β content in the spleen. This indicates that the rats in groups W1, W2, W3, and W4 were more obese, had increased splenic inflammation levels, and experienced more severe loose stools. This further demonstrates that changes in fermentation time affect the quality of Bifidobacterium plantBase-931 fermented products. The quality of fermented products from groups W1, W2, W3, and W4 was lower than that from group Q, and they were not as effective in reducing weight, repairing spleen damage, reducing splenic inflammation levels, and improving loose stools.

[0159] Example 6: The effect of changes in drying methods on fermented products

[0160] 1. Comparative Example 10: The preparation method of Comparative Example 10 is the same as that of Example 1, except that the inlet air temperature for spray drying in Comparative Example 10 is different. Specifically, step 8 of Comparative Example 10 is modified to "spray dry the supernatant B, with an inlet air temperature of 200°C, an inlet liquid volume flow rate of 2 mL / min, and an air velocity of 3 m / min". 3 The air pressure was 0.1 MPa, and the powder was collected as raw material powder No. 1.

[0161] 2. Comparative Example 11: The preparation method of Comparative Example 11 is the same as that of Example 1, except that the freeze-drying process of Comparative Example 11 is different. Specifically, step 10 of Comparative Example 11 is modified to: "Add precipitate A to the No. 2 material solution and stir evenly. Place it in a vacuum freeze dryer for freeze-drying. That is, lower the temperature of the plate to -4°C within 10 min and then lower it to -45.5°C for pre-freezing, and maintain it for 1 h. During the first drying, heat the plate to -30°C within 1 h and run it under a vacuum of 20 Pa for 18 h. During the second drying, control the temperature of the plate to heat to 35°C within 1 h and run it under a vacuum of 2 Pa for 16 h to obtain the freeze-dried raw material. Grind the freeze-dried raw material to 80 mesh using a pulverizer and collect the powder as the No. 2 raw material powder."

[0162] 3. The content of vitamin B6 (calculated as pyridoxine) in the fermented products of Bifidobacterium bifidum PlantBase-931 of Comparative Example 10 and Comparative Example 11 was determined according to the detection method of GB 5009.154. The results are shown in Table 8.

[0163] 4. According to the method of GB 4789.34, the viable count (CFU / g) of Bifidobacterium bifidum PlantBase-931 in the fermented products of Example 1, Comparative Example 10 and Comparative Example 11 was detected. Each sample was tested in triplicate, and the average value was calculated. The results are shown in Table 9.

[0164] 5. Thirty-two obese adult males, aged 25–55 years, with a BMI of 30 ≤ BMI < 34.9, and having had at least 4 episodes of loose stools in the past week, were recruited as subjects. The subjects were randomly divided into four groups of eight, designated X1, X2, X3, and X4. Group X1 consumed 100 mL of 37°C warm water; Group X2 consumed 3g of the fermented product from Example 1; Group X3 consumed 3g of the fermented product from Comparative Example 10; and Group X4 consumed 3g of the fermented product from Comparative Example 11. All consumption was carried out half an hour after lunch each day for 21 consecutive days. The fermented product was prepared by mixing it with 100 mL of 37°C warm water and drinking it directly. On day 16, the number of loose stools over the seven days was recorded, and the average value for each group was calculated. The results are shown in Table 10.

[0165] 6. Results and Analysis

[0166] (1) Detection of Vitamin B6 and Live Bacteria

[0167]

[0168] As shown in Tables 6 and 8, compared with Example 1, the content of vitamin B6 (calculated as pyridoxine) in the fermented product of Comparative Example 10 was significantly reduced, indicating that the change in the inlet air temperature of spray drying has an impact on the quality of the fermented product. This further shows that the quality of the fermented product of Comparative Example 10 is lower than that of the fermented product of Example 1, and it cannot effectively remove dampness and promote weight loss.

[0169]

[0170] As shown in Table 9, compared with Example 1, the viable count of Bifidobacterium bifidum PlantBase-931 in the fermented product of Comparative Example 11 was significantly reduced, indicating that the change in the freeze-drying process affected the quality of the fermented product. This further demonstrates that the quality of the fermented product of Comparative Example 11 was lower than that of the fermented product of Example 1, and it could not effectively remove dampness and promote weight loss. Combining Tables 6, 8, and 9, it can be seen that the drying method in Example 1 was the optimal drying method, resulting in the highest content of vitamin B6 and the highest viable count of Bifidobacterium bifidum PlantBase-931 in the obtained fermented product.

[0171] (2) Population test results

[0172]

[0173] As shown in Table 10, compared with group X1, the number of times the subjects in groups X2 to X4 had loose stools decreased, with different degrees of decrease, indicating that the change in drying method affected the quality of the fermented product. Among them, the subjects in group X2 had the lowest number of loose stools, indicating that the improvement effect on the loose stool condition of the subjects was the best, further demonstrating that the fermented product of Example 1 is better at improving the loose stool symptoms of obese people.

[0174] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Bifidobacterium bifidum PlantBase-931, a herb for removing dampness and aiding weight loss, is characterized by: The preservation number of the Bifidobacterium plantBase-931 is GDMCC No. 66870.

2. The Bifidobacterium bifidum PlantBase-931 according to claim 1, characterized in that, The Bifidobacterium plantBase-931 exerts its effect of removing dampness and reducing weight from at least one of the following (1) to (8): (1) Increase blood GAS and MTL levels, and strengthen the spleen and stomach; (2) Weight loss and weight gain; (3) Increase the relative mRNA expression levels of AQP3 and AQP4 in colon tissue and accelerate the excretion of moisture; (4) Increase the polypeptide content in fermented products; (5) Increase the content of antioxidant peptides in fermented products; (6) Improve the DPPH∙free radical scavenging rate of fermented products; (7) improving ABTS of a fermented product + radical scavenging rate; (8) Increase the vitamin B6 content in fermented products.

3. A fermented product made from Bifidobacterium bifidum PlantBase-931, which is used for removing dampness and aiding weight loss. Its characteristics are: The composition is prepared by using the Bifidobacterium plantBase-931 fermentation composition as described in claim 1 or 2, followed by processing such as centrifugation, spray drying, vacuum freeze drying, pulverization, and mixing. The composition consists of the following materials in parts by weight: 10-20 parts navel orange, 1-10 parts hawthorn, 10-20 parts grape, 1-10 parts lychee, 5-15 parts papaya, 1-10 parts coix seed, 1-10 parts winter melon, 5-15 parts white sugar, and 42 parts purified water.

4. The Bifidobacterium bifidum PlantBase-931 fermented product according to claim 3, characterized in that, The composition comprises the following ingredients in parts by weight: 15 parts navel orange, 5 parts hawthorn, 10 parts grape, 5 parts lychee, 8 parts papaya, 3 parts coix seed, 5 parts winter melon, 7 parts white sugar, and 42 parts purified water.

5. A method for preparing the Bifidobacterium plantBase-931 fermentation product as described in claim 3 or 4, characterized in that, The preparation method includes the following steps: After soaking, decocting and filtering, coix seed water is obtained. Navel orange, papaya, hawthorn, grape, winter melon and lychee are placed in a high-speed blender, and then white sugar and purified water are added. After blending, the filtrate is filtered out. The filtrate is then mixed with coix seed water and sterilized. After cooling, No. 1 liquid is obtained. Bifidobacterium bifidum PlantBase-931 was activated to prepare a seed culture medium; the seed culture medium was added to the No. 1 material solution for staged fermentation, centrifugation, spray drying, vacuum freeze drying, pulverization and mixing to obtain the fermented product.

6. The method for preparing the Bifidobacterium plantBase-931 fermentation product according to claim 5, characterized in that, The phased fermentation includes: the first stage fermentation method is to add 4 parts of seed culture medium to the No. 1 material solution and anaerobic culture at 37°C for 16 hours; the second stage fermentation method is to add 6 parts of seed culture medium to the No. 1 material solution and anaerobic culture at 37°C for 8 hours. The spray drying process includes: an inlet air temperature of 165°C; The vacuum freeze drying process includes heating the shelf to 25°C within 1 hour during the second drying process.

7. The application of the *Bifidobacterium bifidum* PlantBase-931 as described in claim 1 or 2, or the *Bifidobacterium bifidum* PlantBase-931 fermented product as described in any one of claims 3-4, or the *Bifidobacterium bifidum* PlantBase-931 fermented product obtained by any one of the preparation methods in claims 5-6, characterized in that, Application in at least one of the following (1) to (9): (1) Application in the preparation of products for removing dampness and losing weight; (2) Application in the preparation of dehumidifying products; (3) Application in the preparation of weight loss products; (4) Application in the preparation of products for strengthening the spleen and stomach; (5) Application in the preparation of products that increase blood GAS and MTL levels; (6) Application in the preparation of products that increase the relative mRNA expression levels of AQP3 and AQP4 in colon tissue; (7) Application in the preparation of products that reduce IL-1β inflammation levels in the spleen; (8) Application in the preparation of products for repairing spleen damage; (9) Application in the preparation of products that reduce the frequency of loose stools.

8. The application according to claim 7, characterized in that, The applications include live bacteria and inactivated bacteria.

9. The application according to claim 7, characterized in that, The products include solid, liquid, and semi-solid types.

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